SIK2 or -3 silencing by CRISPR/Cas9 mediated gene editing reduces osteoclast differentiation and impairs resorption activity upon RANKL stimulation.

<p><b>A)</b> RAW264.7 cells were infected with pLentiCRISPR-V2 viruses targeting SIK1, SIK2, and SIK3, respectively as detailed in <i>Material and Methods</i> and SIK expression was determined in cell lysates (25 μg/lane) form RAW 264.7 infected cells or wild type cells separated by 7.5% PAGE-SDS and probed with specific antibodies and anti-tubulin. A representative example is depicted (n = 3). <b>B</b>) SIK KO or wild type RAW cells were incubated with 50 ng/ml RANKL for 4 days and then were fixed and stained with TRAP. The TRAP positive multinucleated cells (TRAP+ MNCs) were visualized by digital microphotography and the TRAP+ MNCs containing more than 3 nuclei from four randomly selected magnification fields (scale bar 100 μm) were counted. Error bars = mean +/- SD, from one independent experiment performed in triplicate. <b>C</b>) Cells Lysates obtained from SIK KO cells or wild type RAW 264.7 cells cultured in absence or presence of RANKL (50 ng/ml) for 24 h were separated by 7.5% PAGE-SDS and SIK expression determined as above (<b>i</b>), in the same lysates separated on 10% PAGE-SDS (50 μg/lane) the expression of c-Fos and NFATC1 were determined (<b>ii</b>). GAPDH expression was used as loading control. A representative example (n = 3) is depicted. <b>D)</b> SIK KO cell or wild type RAW were cultured as above in 24 well osteo plates. After 4 days the cells were removed with 5% sodium hypochlorite solution and resorbed areas were measured. Four randomly selected magnification fields (scale bar 100 μm) for each condition were analyzed. The histograms represent the relative resorbed area (%) compared with that of wild type RAW or SIK1 control (set = 100%). Data are expressed as mean +/- SD, from one experiment performed in triplicate. Data are representative of two independent experiments. <i>Insets</i> show a representative example for each condition. * P<0.05, **P<0.01, ***P<0.001.